All microorganisms must assimilate nitrogen from their environment, including microbial pathogens that can use amino acids as sources of nitrogen, energy, and carbon. Nitrogen regulator I (NRI) is a transcription activator in Escherichia coli that coordinates the bacterial response to nitrogen deprivation, the nitrogen regulated (Ntr) response. NRI-dependent activation of gene expression is surprisingly complex - it involves enhancer-like binding sites that can be 1000 base pairs from the start site of transcription, phosphorylation of NRI, two components of cooperative binding to DNA (phosphorylation-independent and phosphorylation-enhanced) that involves two different domains, and a direct contact between NRI and RNA polymerase. Because of features unique to NRI-dependent transcription activation, such activation could provide fundamental insights into mechanisms of gene regulation and signal transduction in both prokaryotes and eukaryotes. For example, the synthesis of virulence factors of microbial pathogens often involves control by proteins homologous to the regulators of the Ntr response. The long-range goals are to thoroughly analyze the mechanism of transcriptional activation and to examine how variations in this mechanism can coordinate the expression of genes of the Ntr regulon. The first specific aim is to determine the structural determinants and function of a component of cooperative binding to DNA that is mediated by the highly conserved, phosphorylated N-terminal domain of NRI. Mutants specifically defective in this form of dimer-dimer interactions will be isolated and the effects on expression of various Ntr genes will be examined. The second specific aim is to characterize one specific step in open complex formation, the interaction between NRI and RNA polymerase. Two biochemical assays will be developed to analyze this interaction; then, the form of NRI (phosphorylated or phosphate-free, bound to a nucleotide or nucleotide-free, or dimeric or tetrameric) that interacts with RNA polymerase will be determined. Finally, the hypothesis that a particular conserved region of NRI is required for the interaction with RNA polymerase will also be tested by mutagenesis of this region. The third aim is to analyze the defect in novel glnG (NRI-encoding) mutants in which some Ntr genes are not expressed, but others are expressed normally. The genes that are not expressed in the mutants have an unusual extra layer of regulation in a wild-type background that the expressed genes do not have. The novelty of these mutants is that they suggest that two different sets of environmental stimuli can control the activity of one transcriptional regulator and that each stimulus controls a different set of NRI-dependent genes.